1. Field of the Invention
This invention relates to the characterization of the role that glycosylation of the transmembrane glycoprotein E2 of highly virulent Classical Swine Fever Virus (CSFV) strain Brescia plays during infection in the natural host and to the utilization of a strategy for manipulating the pattern of glycosylation for particular E2 glycosylation sites in order to alter CSFV virulence, providing a useful tool in the design and development of CSF live-attenuated vaccines.
2. Description of the Relevant Art
Classical swine fever (CSF) is a highly contagious disease of swine. The etiological agent, CSF virus (CSFV), is a small, enveloped virus with a positive, single-stranded RNA genome and, along with Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV), is classified as a member of the genus Pestivirus within the family Flaviridae (Becher et al. 2003. Virology 311: 96-104). The 12.5 kb CSFV genome contains a single open reading frame that encodes a 3898-amino-acid polyprotein and ultimately yields 11 to 12 final cleavage products (NH2-Npro-C-Ems-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) through co- and post-translational processing of the polyprotein by cellular and viral proteases (Rice, C. M. 1996. In: Fundamental Virology, 3rd edition, Knipe et al., eds., Lippincott Raven, Philadelphia, Pa., pages 931-959).
Structural components of the CSFV virion include, the capsid (C) protein and the glycoproteins Ems (E0), E1, and E2. The glycoproteins E1 and E2 are anchored to the envelope at their carboxyl termini. Ems is also referred to as E0; E0 loosely associates with the viral envelope (Thiel et al. 1991. J. Virol. 65: 4705-4712; Weiland et al., 1990. J. Virol. 64: 3563-3569; Weiland et al. 1999. J. Gen. Virol. 80: 1157-1165). E1 and E2 are type I transmembrane proteins with an N-terminal ectodomain and a C-terminal hydrophobic anchor (Thiel et al, supra). E2 is considered essential for CSFV replication, as virus mutants containing partial or complete deletions of the E2 gene have proven non-viable (van Gennip et al. 2002. Vaccine 20: 1544-1556). E2 is the most immunogenic of the CSFV glycoproteins (Konig et al. 1995. J. Virol. 69: 6479-6486; van Gennip et al. 2000. Vaccine 19: 447-449; Weiland et al. 1990, supra), inducing neutralizing antibodies and protection against lethal CSFV challenge. E2 has been implicated, along with E0 (Hulst and Moormann. 1997. J. Gen. Virol. 78:2779-2787) and E1 (Wang et al. 2004. Virology 330:332-341), in viral adsorption to host cells; indeed, chimeric pestiviruses exhibit infectivity and cell tropism phenotypes consistent with those of the E2 gene donor (Liang et al. 2003. J. Gen. Virol. 84:1269-1274; van Gennip et al. 2000, supra). Modifications introduced into these glycoproteins appear to have an important effect on CSFV virulence (Meyers et al. 1999. J. Virol. 73: 10224-10235; Risatti et al., 2005a, J. Virol. 79: 3787-3796; Risatti et al. 2005b, Virology 343: 116-127; van Gennip et al. 2004. J. Virol. 78: 8812-8823).
Glycosylation is one of the most common types of protein modifications. N-linked oligosaccharides are added to specific asparagine residues in the context of the consensus sequence Asn-X-Ser/Thr (Kornfeld and Kornfeld. 1985. Annu. Rev. Biochem. 54: 631-664). Intracellular O-glycosylation is characterized by the addition of N-acetylglucosamine to serine and threonine residues in a protein, although the acceptor site does not display a definite consensus sequence (Gupta and Brunak. 2002. Pac. Symp. Biocomput. 310-322). Not all predicted sites in a protein sequence are used for carbohydrates, since many of them are inefficiently glycosylated (Shakin-Eshleman et al. 1992. J. Biol. Chem. 267: 10690-10698) or remain unglycosylated (Gavel and von Hejne. 1990. Prot. Eng. 3:433-442).
Putative N-glycosylation sites within CSFV E2 have been predicted previously (Moormann et al., 1990. Vet Microbiol. 23: 185-191; van Rijn et al. 1994. J. Virol. 68: 3934-3942). According to a glycosylation analysis algorithm (http://www.cbs.dtu.dk/services/), E2 of the CSFV strain Brescia has five putative N-linked and one putative O-linked glycosylation sites, although this is not confirmed by experimental evidence. A sixth N-linked glycosylation site is present in several CSFV strains, with the Brescia sequence differing in one amino acid from the consensus (Asn-X-Ser/Thr). Predicted E2 glycosylation sites are highly conserved among CSFV isolates. Even though glycosylation of E0, E1, or E2 proteins may play a significant role in the CSFV viral replication cycle, the function of added oligosaccharides is not known. In general, glycosylation of enveloped virus structural proteins has been shown to be important for receptor binding, membrane fusion, penetration, virus budding, and infectivity as analyzed in cultured cells (Abe et al. 2004. J. Virol. 78: 9605-9611; Doms et al. 1993. Virology 193: 545-562; Hanna et al. 2005. J. Virol. 79: 13262-13274; Shi et al. 2005. J. Virol. 79: 13725-13734; Shi and Elliott. 2004. J. Virol. 78: 5414-5422). However, the significance of viral envelope protein glycosylation in virus replication, pathogenesis, and virulence in the natural host is unknown. Recently, it has been shown that glycosylation of Porcine Respiratory and Reproductive Syndrome virus (PRRSV) GP5 affects virus infectivity, antigenicity and ability to induce neutralizing antibodies in swine (Ansari et al. 2006. J. Virol. 80: 3994-4004). Loss of N-linked glycosylation from the hemagglutinin-neuraminidase protein from Newcastle Disease Virus, a bird pathogen, results in attenuation of the virus in chickens (Panda et al. 2004. J. Virol. 78: 4965-4975). Similarly, degrees of virulence in chickens have been associated with glycosylation patterns of surface proteins hemagglutinin and neuraminidase of highly pathogenic avian influenza virus H5N1 (Hulse et al. 2004. J. Virol. 78: 9954-9964). Pathogenic phenotypes observed upon infection of natural hosts with modified viruses link glycosylation of virus surface proteins with mechanisms such as evasion of the immune system in PRRSV (Ansari et al., supra), attenuation in NDV (Panda et al., supra), and determinants of virulence in avian influenza (Hulse et al, supra).
Strategies for controlling disease in the event of a CSFV outbreak include the production of rationally designed live attenuated vaccine CSFV strains. Thus, the effect of modification of glycosylation sites of other of the CSFV virion glycoproteins need to be evaluated. Here, we report the effects of modification of particular E2 glycosylation sites. We used oligonucleotide site-directed mutagenesis of the E2 gene of the highly virulent CSFV strain Brescia to construct a panel of glycosylation mutants. These mutants were evaluated to determine whether the removal of each of these glycosylation sites in the E2 glycoprotein could affect viral infectivity and virulence in swine.